Leukocytes in the peripheral blood of normal subjects consist of five types, i.e., lymphocytes, monocytes, neutrophils, eosinophils and basophils. The latter three kinds of leukocytes are collectively referred to as granulocytes. Different leukocyte types have different functions and counting of leukocytes in the blood according to their type provides valuable information for diagnostic purposes. For instance, an increase in the number of neutrophils is associated with such diseases as inflammations, myocardial infarction and leukemia, and a decrease in their number is associated with viral diseases, hypoplastic anemia, agranulocytosis, etc. On the other hand, an increase in the number of eosinophils is found in such diseases as parasitosis, Hodgkin's disease and allergosis. An increased number of monocytes occurs either during the convalescence period of patients suffering from infections diseases or in such diseases as monocytic leukemia.
In addition to the above-mentioned lymphocytes, monocytes, neutrophils, eosinophils and basophils which are generally referred to as "normal cells", abnormal cells occasionally appear in the peripheral blood of patients suffering from certain hemodyscrasias. For instance, gemmules or blasts are sometimes found in the peripheral blood of leukemia patients. Such abnormal cells also occur in various types and classifying them and determining the number of cells in each class of abnormal cells is also of great importance in clinical fields.
The classification and counting of leukocytes has most commonly been conducted by the differential counting method which is also referred to as the visual counting method, or simply as the manual technique.
In this method, a blood sample is smeared on a glass plate and the blood corpuscles in the smear are stained for examination by microscopy. The technician identifies the type of individual leukocytes according to their morphological features (e.g., their size, the morphology of their nucleus and cytoplasm, and the presence or absence of granules) or the degree of dye uptake and performs classification and counting of them. At ordinary laboratories, 100-200 leukocytes are usually counted for each sample and the percentage of the total leukocyte count occupied by each type of corpuscle is recorded as a measured value.
The differential counting method has several disadvantages. First, microscopic observation must be preceded by cumbersome procedures for preparing a specimen that involve such steps as smearing a blood sample on a glass plate, fixing the corpuscles and staining them. Secondly, it is a great burden for the technician to identify subtle differences between corpuscles by microscopic classification and counting. Thirdly, it is difficult even for a skilled technician to yield consistent counts by the manual method since, aside from the small number of leukocytes computed, the smeared sample often has an uneven distribution of blood corpuscles.
Under these circumstances, there has arisen a strong need for a method to be developed that is capable of automated classification and counting of leukocytes. The automated techniques so far realized may be roughly divided into two types.
The first method consists of recording the images of corpuscles with a video camera or some other suitable imaging device classifying the leukocytes by means of image processing on a computer. The operating principles of this method are similar to those of the conventional visual counting method but primarily due to the existence of many corpuscles that defy classification by processing with a computer, this method has not yet become an ideal alternative to the manual method. Another problem with this method is that it requires sophisticated equipment which is bulky and costly.
The other approach toward automatic classification and counting of leukocytes is based on a flow system. In this method, a blood sample having corpuscles suspended in a diluent is permitted to flow in such a way that the corpuscles will individually (one by one) pass through a constricted detector and leukocyte classification is conducted by analyzing the signals generated by the detector. This second method which makes use of a flow system is further subdivided into two categories.
In a method of the first category, an electrolyte in which all red cells present are disrupted with a lysing agent so that only leukocytes will be suspended is permitted to flow through an orifice and the change in electrical impedance that occurs at the orifice when each corpuscle passes through it is detected, the magnitude of the detected signal being used as a basis for classification of leukocytes.
A method of the second category is characterized by the use of a flow cytometer that comprises a light source, a flow cell that permits the blood cells in a sample to flow one by one through a constricted channel, a photometric unit that detects light issuing from each blood cell, and an analyzer for analyzing the detected signal. In this method, the corpuscles in the sample which are stained are illuminated under light and the fluorescence emitted from the irradiated corpuscles is detected, optionally together with scattered light, with leukocyte classification being conducted in accordance with the intensity of the detected signal.
This method of the second category has problems in practical use such as the need to adopt a complicated staining process, as well as the use of sophisticated and costly equipment including an optical system.
One of the principles underlying the method of the first category is disclosed in Japanese Patent No. 508,789 and U.S. Pat. No. 3,390,326. According to this principle, a sample prepared by suspending particles in a fluid medium having a different dielectric constant is allowed to pass through a fluid channel having a constricted portion held between closely adjacent electrodes and the change that occurs in the electric impedance between the electrodes on account of the difference in dielectric constant between the particles and the fluid medium is detected.
A description of a practical apparatus that operates on this principle may be found in Ichiro Kurokawa et al., "Toa jidokekkyukeisuki no shiyoukeiken (Experience of the Use of Toa Automatic Blood Cell Counter)" in "Rinsho Byori (Clinical Pathology)", vol 16 pp 251-255, 1968. This apparatus has a 3.5 MHz high-frequency oscillator which applies a high-frequency current to the detector circuit, and the change in electric impedance that occurs between the electrodes in the detector circuit as corpuscles in suspension pass through the detector circuit is detected.
A description of leukocyte measurements with a Toa Automatic Blood Cell Counter may be found in Ichiro Kurokawa et al., "Rinsho Kensa (Clinical Testing)" vol. 11, pp. 148-151, 1967. The described method consists of adding saponin to a suspension of blood cells so that the erythrocytes are lysed to enable the measurement of leukocytes that are left intact.
Besides saponin, there are several other lysing agents available that are capable of lysing erythrocytes. Among such blood lysing agents, CTAC (cetyltrimethyl ammonium chloride) and Tergitol monionic NPX display a strong lytic activity under use conditions but, at the same time, the protoplasm of leukocytes is also attacked and their nuclei will become almost naked. In contrast, saponin allows leukocytes to remain fairly close to their intact state. Therefore, CTAC and Tergitol monionic NPX are unsuitable for use as blood lysing agents in a model such as Toa Automatic Red Cell Counter that detects the change in electric impedance at high frequencies (see Kazuo Shintani, "Hakekkyusantei no mondaiten (Problems with Leukocyte Counting)" in "Rinsho Kensa (Clinical Testing)" vol 12, pp 900-905, 1968.
In determining the number of leukocytes with an automatic blood cell counter, it is necessary that the magnitude of signals from leukocytes be sufficiently large compared to the signals of dissolved erythrocyte membranes (ghosts) and attendant noise to enable clear differentiation between the two kinds of signals. In order to determine whether this condition is established, a cumulative size-frequency distribution curve as shown in FIG. 15 is often used. The graph shown in FIG. 15 is constructed by plotting the signal threshold values of an automatic blood cell counter on the horizontal axis, and the number of detected signals exceeding a certain signal threshold value on the vertical axis. In FIG. 15, portion A is generally referred to as "a flat portion" of the cumulative size-frequency distribution curve, and in order to ensure that leukocyte signals being measured are sufficiently larger than the above-mentioned noise and ghost signals to yield consistent leukocyte counts, the equipment and reagents used therewith must be adjusted so as to allow the flat portion A to be extended.
In practice, however, it has been pointed out that measurements of leukocytes with a Toa Automatic Blood Cell Counter using saponin as a blood lysing agent produce a shorter "flat portion" than when the DC method to be described later in this specification is employed. For instance, the graph in FIG. 15 was constructed from the data in Table 1 given in Yuji Takamori et al., "Hakekkyuryudobunpu no hendoyoin to sono keisuchi ni oyobosu eikyo ni tsuite (On Factors Causing Variations in Leukocyte Size-Frequency Distribution and Their Effects on Leukocyte Counts)" in MCC News, No. 34, pp. 12-21, Toa Tokushu Denki, Hyogo, Japan, 1969 (the data shows the cumulative size-frequency distribution obtained with saponin added to samples left for 5 minutes after dilution), and the flat portion of the curve in this graph extends only from the threshold value of 350 to 400.
As for the shortness of the flat portion obtained when leukocyte measurements are conducted with a Toa Automatic Red Cell Counter using saponin, P. W. Helleman, et al., Scand. J. Haematology, 6 pp. 160-165, 1969, comments that this phenomenon would be due to the difference in dielectric properties between dissimilar types of leukocytes (as between a lymphocyte and a granulocyte).
A careful review of the cumulative size-frequency distribution curve in FIG. 15 will show that it contains a second flat portion B on the right side of the first flat portion A. This fact will become clearer if one constructs a size-frequency distribution curve as shown in FIG. 16 by plotting the number of leukocytes for each threshold level as calculated from the cumulative size-frequency distribution curve in FIG. 15. Stated more specifically, FIG. 16 contains the population of erythrocyte ghosts and noise which is indicated by C, the first population of leukocytes indicated by D which lies rightward of C, and the second population of leukocytes indicated by E which is situated rightward of D. It is therefore possible to say that the division of the leukocyte size-frequency distribution into two populations has produced short flat portions in the cumulative size-frequency distribution.
The foregoing discussion amounts to a showing of the fact that two different populations of leukocytes have to date been classified and counted by the Toa Automatic Blood Cell Counter and that the above-described method of detecting the change in electric impedance at high frequency (This method is hereinafter referred as the RF method) has the potential to count leukocytes after classifying and differentiating them into several types.
However, at the time when the Toa Automatic Blood Cell Counter was developed and commercialized, the primary concern was to obtain leukocyte counts in a consistent and reliable manner and no detailed investigation was conducted to unravel the reason for the shortness of flat potions of a cumulative size-frequency distribution curve. On the contrary, most efforts were directed at adjusting the equipment and reagents in such a way that the flat portions could be extended as much as possible to ensure consistent counting of leukocytes.
Besides the RF method described above, the principle underlying the method of the first category which is included in the scope of the methods making use of a flow system is also described in Japanese Patent No. 217,947 and U.S. Pat. No. 2,656,508. According to this principle, a sample having particles suspended in a fluid medium having a different conductivity is allowed to pass through a narrow current channel and any change in current that occurs on account of the difference in conductivity between the particles and the fluid medium is detected. This method is hereunder referred to as the DC method. In the DC method, the magnitude of a signal detected is substantially proportional to the volume of particles.
When this DC method is combined with the already-described RF method, information on the volume of particles is obtained by the DC method and, in addition to that, information derived from the structure of the particles and the properties of the constituent materials of the particles can be obtained. An apparatus that relies on this approach for classifying several populations of different types of particles from a system comprising a mixture of different types of particles in the same suspension is disclosed in Japanese Patent No. 785,859 and U.S. Pat. No. 3,502,974. However, these patents show nothing about the possibility of classifying and counting different populations of leukocytes.
The already-described RF method may be modified in such a way that, instead of disrupting corpuscles, their contents are replaced so that their dielectric constant is changed, followed by classification and counting procedures. A method based On this approach is disclosed in Japanese Patent No. 936,823 and U.S. Pat. No. 3,836,849. In the second example of these patents, 250 .mu.l of a 1% saponin solution is added to 100 .mu.l of whole blood suspended in a phosphate buffered physiological saline solution having a pH of 7.2, and then the erythrocytes are lysed and two distinct peaks for leukocytes appear. This result agrees well with that obtained by performing measurements with a Toa Automatic Blood Cell Counter (FIG. 16).
Even if the RF method is performed using saponin which produces a comparatively mild action on corpuscles, the protoplasm of leukocytes is slowly disrupted to cause gradual attenuation of signals from leukocytes. If one wants to replace the contents of corpuscles at normal pH's without disrupting them as in Japanese Patent No. 936,823, it is necessary to reduce the concentration of saponin to a significantly low level so that it will act slowly on leukocytes. However, in this case, the preliminary treatment for starting a measurement takes quite a long time as compared to the time of the preliminary treatment necessary for ordinary leukocyte measurements (3-5 minutes), so it is not suitable for practical purposes to use this method with automatic analyzers which have to process many specimens in a short period of time.
A different approach that brings about a change in leukocytes in a fairly short period of time and which classifies the leukocytes into three populations on the basis of the amount of that change has been realized using the DC method and is described in National Publication of Translated Version No. 500097/1985 and U.S. Pat. No. 4,485,175.
In this method, a quaternary ammonium salt which is also described in Japanese Patent No..936,823 is used as a cytolytic agent and by using it at low concentration, a change in the volume of leukocytes is produced and the leukocytes are classified into three populations on the basis of this difference. However, the results of an example given in U.S. Pat. No. 4,485,175 are as shown in FIG. 17 and lymphocytes, monocytes and granulocytes in the three populations of leukocytes are not necessarily completely separated or differentiated.
Furthermore, the quaternary ammonium salt, even if it is used at low concentration, has such a great effect on corpuscles that hemolysis will take place within a short period of time. According to Japanese Patent No. 936,823, only the dielectric constants of corpuscles are changed without replacing or disrupting their contents, but in the absence of any specific data, it is not clear whether this is actually possible.
Considering the fact that quaternary ammonium salts cause an undesirably high degree of damage to leukocytes that are to be classified and counted, National Publication of Translated Version No. 502277/1986 and International Publication No. WO85/05684 propose a method in which mildly acting saponin is added at high concentration and its lysing action is quenched at the time when the erythrocytes have been lysed and only thereafter is analysis performed with a flow cytometer or by the combination of the Rf and DC methods.
This method requires a fixing agent in order to quench the lysing action and, furthermore, a special procedure such as adding it at a predetermined timing and thereafter heating the cells at elevated temperatures must be taken. This method is therefore not very effective for use with automatic analyzers.
As described above, among the methods that automatically classify and count leukocytes using a flow system, the method of the second category which makes use of flow cytometer has a disadvantage in that the equipment is sophisticated and expensive. As for the method of the first category which detects that change in impedance that occurs at an orifice when corpuscles pass through the orifice, and wherein leukocytes are classified in accordance with the magnitude of detected signals, certain problems also arise depending on whether leukocytes are changed mildly or violently: in the former case, it takes as much time to complete the preliminary treatments or a special fixing agent must be added at a predetermined timing or subsequent heat treatment is necessary; in the latter case, leukocytes are damaged so extensively that they cannot be classified into more than three types. Therefore, none of the methods of the second category that have so far been proposed are satisfactory for practical purposes. As for the method of the first category, no technique has ever existed that is capable of classifying and counting abnormal cells such as the already-described blasts by utilizing this method.
Furthermore, saponin as a cytolytic agent to be used in the method of the first category is labile in terms of blood lysing action whereas quaternary ammonium salts are too violent as noted above.
According to the first embodiment of the present invention, neither saponin nor quaternary ammonium salts are used as cytolytic agents, and instead a cytolytic agent having consistent lysing action is used to lyse erythrocytes and damage leukocytes in a very short period of time. Thereafter, analysis is made by the combination of the already-described DC and RF methods, thereby providing remarkable advantages, such as classification of normal cells into five types as well as classification of abnormal cells, which have been unattainable in the prior art techniques of leukocyte classification.
On the other hand, Japanese Patent Public Disclosure No. 134957/88 (EP Patent Publication No. 259833) discloses a process for classifying leukocytes (a method of the second category) comprising hemolyzing erythrocytes in a first solution which is acidic and hypoosmotic, neutralizing the acidity of the first solution and adjusting the osmolarity by using a second solution, and measuring leukocytes by a flow cytometer to classify them. Moreover, Patent Domestic Announcement No. 502931/89 (PCT/US88/00762) discloses a process (a method of the first category) for classifying leukocytes comprising lysing erythrocytes in a first solution which is acidic and hypoosmotic, neutralizing the acidity of the first solution and adjusting the osmolarity by using a second solution, and classifying and counting leukocytes by using the RF and DC methods in combination.
However, ghosts due to erythrocytes (hereinafter referred to as "erythrocyte ghosts") cannot be sufficiently reduced only by hemolyzing erythrocytes in an acidic and hypoosmotic as conducted in the above two methods. Therefore when measuring leukocytes by using RF and DC methods, said ghosts and the intensity of the detected signs partially overlap and as a result, cannot be definitely distinguished. Since either of these two methods requires a reagent consisting of two solutions, an automatic blood analyzer wherein such methods are practiced necessarily becomes complicated.
Incidentally, although it is described in a reference [Clinical Analysis (Rinsh6 Kensa) Book, Additional Volume 1, "Pretreatment for Examination of Samples", page 3 published by Kanahara Shuppan] that a smear must be prepared within 4 hours after blood gathering, smears are often prepared many hours after blood gathering in a big institution such as a clinical examination center. In such cases, injury of leukocytes increases as time passes and particularly neutrophils and monocytes are remarkably injured. Therefore, results of the classification of leukocytes obtained from blood gathered 24 hours previously are largely different from those of leukocytes obtained immediately after blood gathering.
Therefore there has been a need for a method for accurately classifying leukocytes in blood gathered many hours earlier.
The object of the second embodiment of this invention is to provide a process wherein neither saponin nor quaternary ammonium is used as a cytolytic agent but another cytolytic agent exhibiting stable hemolytic activities is used to hemolyze erythrocytes and to stabilize leukocytes in a short time, wherein erythrocyte ghosts can be distinguished from lymphocytes and normal leukocytes are classified into five types and abnormal cells are classified, and wherein accurate values of the classified leukocytes can be obtained for blood gathered many hours earlier.